Immunoassays of various types have become the methods of choice for quantitating many substances of interest in biological fluids. All of the assays are quite similar in principle, but differ in the method for quantitating the amount of labelled substance bound to the analyte of interest.
In radioimmunoassay (RIA) either purified antigen or its specific antibody is labelled with a radioisotope, such as iodine-125 (I.sup.125). A diagrammatic representation of the reaction mechanisms involved in the two main types of RIA are shown below.
______________________________________ COMPETITIVE PROTEIN BINDING ASSAY ##STR1## TWO SITE IMMUNORADIOMETRIC ASSAY ##STR2## ##STR3## ______________________________________ Key: ##STR4## Ab* = labelled antibody; Ag* = labelled analyte.
In the competitive assay labelled and unlabelled analyte compete for binding sites on the antibody attached to the solid phase. After incubating, the solid phase is separated from the reaction mixture, and washed to remove residual unbound radioactivity. The amount of radioactivity bound to the solid phase is determined in a gamma counter. A standard curve is prepared using calibrator sera containing known amounts of analyte, and these are run simultaneously with the unknowns. A curve is generated and the unknowns are quantitated from the standard curve. In this case the amount of analyte in the unknown is essentially inversely proportional to the amount of radioactivity bound to the solid phase.
In the two site assay the analyte binds to the antibody attached to the solid phase. The solid phase is washed to remove residual unbound analyte and labelled antibody is added. During incubation the labelled antibody attaches to the analyte that is bound to the solid phase antibody. After incubation, the solid phase is washed to remove excess labelled antibody and the amount of radioactivity in the solid phase is determined. A standard curve is prepared using calibrator sera containing known amounts of analyte, and these are run simultaneously with the unknowns. A curve is generated and the unknowns are quantitated from the standard curve. In this case the amount of analyte in the unknown is essentially directly proportional to the amount of radioactivity bound to the solid phase.
A difficulty with these procedures is the limited assay range. That is, in the two site assay the standard curve only extends about two orders of magnitude, and in the competitive system two to three orders of magnitude. This phenomenon occurs because of the limited binding capacity of the solid phase antibody, and also the affinity constants of the antibody. Monoclonal antibodies have expanded the dynamic range of the assays to some extent, but many substances still require substantial dilution before they can be assayed in these systems.
Enzymeimmunoassays (EIA) operate in quite the same manner as RIA, except that an enzyme is used as a label instead of a radioisotope. Enzymes such as horseradish peroxidase, alkaline phosphatase, glucose oxidase and agalactosidase have been used as labels for antigens and antibodies in EIA systems. The reaction scheme below demonstrates a two site EIA using alkaline phosphatase (E) as a label.
______________________________________ TWO SITE EIA ______________________________________ ##STR5## ##STR6## ##STR7## ______________________________________
From the reaction sequence it can be seen that after binding of the enzyme-labelled antibody, the solid phase is washed to remove excess labelled antibody and is reacted with a substrate (PNPP) that the enzyme will degrade into a colored compound (PNP). The reaction is stopped by adding a chemical to poison the system and the amount of color is determined spectrophotometrically at a given wavelength. In this case the intensity of the color is directly proportional to the amount of analyte present in the sample in relation to a standard curve, as shown previously.
Fluoroimmunoassays (FIA) in many cases operate on the same basic principle as RIA and EIA, except that fluorescent compounds such as fluorescein, rhodamine and umbelliferone are used as labels. There are other types of FIA that operate on very different principles than those described above, since no solid phase is necessary. Examples of these types of assays are fluorescence polarization and fluorescence quenching.
There are several common difficulties inherent in RIA, EIA and FIA systems. One problem involves the limited dynamic range of the assays, as has been previously discussed. This limitation necessitates preparing multiple dilutions of each sample, as well as, a four-point standard curve. In addition these systems in many cases involve long incubation periods and several wash and reagent addition steps. All of these problems contribute to making the assays tedious, labortensive, time-consuming and costly in terms of reagent use.
All of the foregoing techniques, while sufficient to yield relatively accurate values for the analyte in question, are not entirely satisfactory, because they are either too sensitive, not sensitive enough, too laborious and time consuming or costly in terms of reagents.